標題: Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection
作者: Pan, Kao-Lu
Lee, Jin-Ching
Sung, Hsing-Wen
Chang, Teng-Yuang
Hsu, John T. -A.
生物科技學系
Department of Biological Science and Technology
公開日期: 1-Nov-2009
摘要: A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(Delta 4B5A) SEAP, that carries a dual reporter, EG(Delta 4B5A) SEAP. The EG(Delta 4B5A) SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via Delta 4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG(Delta 4B5A) SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(Delta 4B5A) SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(Delta 4B5A) SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z'-factorof this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.
URI: http://dx.doi.org/10.1128/AAC.00601-09
http://hdl.handle.net/11536/6473
ISSN: 0066-4804
DOI: 10.1128/AAC.00601-09
期刊: ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
Volume: 53
Issue: 11
起始頁: 4825
結束頁: 4834
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